A nucleic acid molecule containing nucleotide sequences that encode the capsid protein, pre-membrane protein and non-structural protein of Japanese encephalitis virus, and a nucleotide sequence that encodes the envelop protein of a second flavivirus, wherein the nucleotide sequence(s) that encode(s) the pre-membrane protein and/or non-structural protein of Japanese encephalitis virus contain(s) nucleotide mutations that produce one or more amino acid mutations that attenuate the virus.

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DESCRIPTION ATTENUATED CHIMERIC FLAVIVIRUS BEARING ATTENUATED JAPANESE
ENCEPHALITIS VIRUS GENE AS BACKBGNE TECHNICAL FIELD
The present invention relates to an attenuated chimeric flavivirus having a gene of attenuated Japanese encephalitis virus as the backbone, which is useful as an attenuated live Vaccine for preventing flavivirus infections.
BACKGROUND ART
Currently, more than about 60 kinds of viruses belonging to Flaviviridae (hereinafter abbreviated flaviviruses) are known, including Japanese encephalitis virus, West Nile virus, dengue 1-4 virus, yellow fever virus, St. Louis encephalitis virus, tick-borne encephalitis virus, Kunjin virus, Central European encephalitis virus, Kyasanur Forest virus, Murray Valley encephalitis virus, Omsk hemorrhagic fever virus, Powassan virus, Russian spring-summer encephalitis virus, Yokose virus, Apoi virus, and Aroa virus.
These flaviviruses have a genome of single-stranded ( + ) RNA and are similar to each other in terms of gene structure. The open reading frame (ORF) of the flavivirus genome encodes three structural proteins (capsid (C) protein, pre-membrane (prM) protein, which is the precursor for membrane (M) protein, and envelop (E) protein) and subsequent seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) from the 5' terminus thereof.
These structural proteins and non-structural proteins of the flaviviruses are translated as Single polyproteins; the polyproteins translated are then processed by the protease and NS3 protein having protease activity of host cells and the virus, resulting in the formation of a mature virion comprising the above-described three kinds of structural proteins.
It is known that many of the above-described flaviviruses infect to mammals including humans, and birds, via insects such

as mosquitoes and ticks, and cause encephalitis and/or febrile Symptoms. Originally, each of these flavivirus species is indigenous to a particular region; therefore, the endemic area of the infection had been limited. However, in recent years, due to development in traffics/distributions, climate change and the like, various flavivirus infections have expanded to places other than the original habitats of causal flaviviruses, posing an important problem with public health.
In preventing the expansion of viral infections, prevention with vaccines is effective. However, despite the fact that a large number of viruses belonging to Flaviviridae have been recognized as described above, only attenuated live vaccines for yellow fever virus and Japanese encephalitis virus infections and inactivated vaccines for Japanese encephalitis virus and tick-borne encephalitis virus infections are in practical application as vaccines for flavivirus infections. Particularly, attenuated live vaccines are useful as vaccines that are inexpensive and induce long-term immunity, but there are no approved live vaccines other than those described above.
In these circumstances, as a means of quickly developing novel attenuated live vaccines for various flavivirus infections, a strategy utilizing a chimeric flavivirus prepared by a gene engineering technique has recently been drawing attention.
For example, ChimeriVax™-JE is a chimeric flavivirus prepared by replacing the genes that encode two structural proteins (prM-E) of the yellow fever virus Vaccine 17D strain with the corresponding genes of the Japanese encephalitis virus Vaccine SA14-14-2 strain (see, for example, pamphlet for International Patent Publication No. 98/37911 and pamphlet for International Patent Publication No. 01/39802) . ChimeriVax™-JE is attenuated to the extent that allows its use as a Vaccine as a result of a plurality of amino acid mutations from the wild type that are present in the virus polyprotein corresponding to

the E protein of the Japanese encephalitis virus Vaccine SA14-14-2 strain (see, for example, Arroyo et al., J. Virol. 75:934-942, 2001).
Furthermore, on the basis of this technique for ChimeriVax™-JE, chimeric flaviviruses with dengue 1-4 viruses (ChimeriVax™-DEN (1-4)) (see, for example, pamphlet for International Patent Publication No. 98/37911 and pamphlet for International Patent Publication No. 01/39802) and a chimeric flavivirus with West Nile virus (ChimeriVax™-West Nile) (see, for example, pamphlet for International Patent Publication No. 2004/045529), which have a gene of the yellow fever virus Vaccine 17D strain as the backbone, have also been developed.
These chimeric flaviviruses are also attenuated to the extent that allows their use as vaccines, with their attenuation resulting mainly from amino acid substitutions from the wild type that are present in the virus polyprotein corresponding to the prM-E protein.
However, it has been reported that the attenuation of ChimeriVax™-DENl results mainly from amino acid mutations in the E protein that occur during the passage of this chimeric flavivirus in cells for Vaccine production (see, for example, Guirakhoo et al., J. Virol. 78:9998-10008, 2004). Furthermore, because re-infection with dengue viruses of different serotypes is likely as a cause of the onset of dengue hemorrhagic fever, this Vaccine has not found a practical application.
Also, ChimeriVax™-West Nile has the attenuation promoted by artificially introducing an amino acid mutation in the E protein derived from the wild-type highly virulent West Nile virus NY-99 strain (see, for example, pamphlet for International Patent Publication No. 2004/045529).
As a chimeric flavivirus using a gene backbone other than yellow fever virus (YF-17D), a chimeric flavivirus prepared by replacing the gene that encodes the prM-E protein of dengue-4 virus with the corresponding gene of the West Nile virus NY99

strain (WN/DEN4 chimeric virus) has been reportecl (see, for example, Pletnev et al., Proc. Natl. Acad. Sei. USA 99:3036-3041, 2002) .
Although the WN/DEN4 chimeric virus is attenuated compared to the parent strains thereof, i.e., West Nile virus and dengue-4 virus, the mechanism of the attenuation has not yet been fully elueidated, and the virus has not found a practical application as a Vaccine.
Disclosure of the Invention
When attenuated live chimeric flavivirus vaccines for various flavivirus infections is developed by conventional methods as described above, it is necessary to investigate the safety, that is, brain neurotoxicity, infectivity from peripheral to the central nervous System (brain nerve invasiveness), infection-preventing effect, neutralizing antibody produetion and the like of the chimeric virus construeted for each combination of flavivirus species, and this may be a major cause of prolonging the period to the practical application of attenuated live Vaccine.
Furthermore, live vaccines still pose the problem of reduetions in antibody produetivity as they are attenuated for the sake of safety. This is because attenuation of viruses by modifying the E protein as described above potentially reduces their immune induetion potential (antibody produetivity) as vaccines because the antigenic determinant for inducing a neutralizing antibody is present in the E protein.
Accordingly, it is an objeet of the present invention to provide an attenuated chimeric flavivirus having an attenuating mutation in a portion other than the E protein.
The present inventors diligently investigated to solve the above-described problem, found that the Japanese encephalitis virus Vaccine ML-17 strain for swine had a plurality of amino aeid mutations intrinsic to the ML-17 strain in the prM and NS proteins other than the E protein, and

obtained the Suggestion that these amino acid mutations might be involved in the attenuation of the ML-17 strain. Based on this finding, the present inventors were inspired to construct a chimeric flavivirus having the structural and non-structural proteins other than the E protein (that is, C protein, prM protein, and NS protein) of a Japanese encephalitis virus comprising one or more of the amino acid mutations intrinsic to the ML-17 strain, conducted further investigations, and developed the present invention.
Accordingly, the present invention provides: [1] A nucleic acid molecule comprising nucleotide sequences that encode the capsid protein, pre-membrane protein and non-structural protein of Japanese encephalitis virus, and a nucleotide sequence that encodes the envelop protein of a second flavivirus,
wherein the nucleotide sequence(s) that encode (s) the pre-membrane protein and/or non-structural protein of Japanese encephalitis virus comprise(s) nucleotide mutations that produce one or more amino acid mutations that attenuate the virus.
[2] The nucleic acid molecule described in [1], wherein the Japanese encephalitis virus is the ML-17 strain. [3] The nucleic acid molecule described in [1] or [2], wherein the second flavivirus is selected from the group consisting of West Nile virus, dengue 1-4 virus, yellow fever virus, St. Louis encephalitis virus, tick-borne encephalitis virus, Kunjin virus, Central European encephalitis virus, Kyasanur Forest virus, Murray Valley encephalitis virus, Omsk hemorrhagic fever virus, Powassan virus, Russian spring-summer encephalitis virus, Yokose virus, Apoi virus, and Aroa virus.
[4] An attenuated chimeric flavivirus encoded by the nucleic acid molecule described in any one of [1] to [3]. [5] An attenuated live Vaccine comprising the attenuated chimeric flavivirus described in [4].

[6] A method of preparing the nucleic acid moiecuie aescrioea in [1], which comprises the following steps: a step for replacing a nucleotide sequence that encodes the envelop protein in a nucleic acid moiecuie comprising a nucleotide sequence that encodes Japanese encephalitis virus with the nucleotide sequence that encodes the envelop protein of the second flavivirus; and
a step for introducing nucleotide mutations that produce one or more amino acid mutations that attenuate the virus into the nucleotide sequence(s) that encode(s) the pre-membrane protein and/or non-structural protein of Japanese encephalitis virus. [7] A method of preparing the nucleic acid moiecuie described in [1], which comprises the following step:
a step for replacing a nucleotide sequence that encodes the envelop protein in a nucleic acid moiecuie comprising a nucleotide sequence that encodes a Japanese encephalitis virus having one or more amino acid mutations that attenuate the virus in the pre-membrane protein and/or non-structural protein with the nucleotide sequence that encodes the envelop protein of the second flavivirus.
[8] A method of preparing an attenuated chimeric flavivirus, which comprises a step for expressing chimeric flavivirus proteins from the nucleic acid moiecuie described in any one of
[I] to [3].
[9] A nucleic acid moiecuie comprising a nucleotide sequence that encodes a Japanese encephalitis virus having one or more amino acid mutations that attenuate the virus in the pre-membrane protein and/or non-structural protein. [10] A vector comprising the nucleic acid moiecuie described in [9].
[II] An attenuated Japanese encephalitis virus encoded by the
nucleic acid moiecuie described in [9]-
[12] A method of preparing the nucleic acid moiecuie described in [9], which comprises a step for introducing nucleotide

mutations that produce one or more amino acid mutations that attenuate the virus into nucleotide sequence(s) that encode(s) the pre-membrane protein and/or non-structural protein in a nucleic acid molecule comprising a nucleotide sequence that encodes Japanese encephalitis virus.
[13] A method of preparing an attenuated Japanese encephalitis virus, which comprises a step for expressing Japanese encephalitis virus proteins from the nucleic acid molecule described in [9].
By the present invention, an attenuated chimeric flavivirus having attenuating mutation(s) in portion(s) other than the E protein is provided. Because the attenuation of the chimeric flavivirus of the present invention can be achieved without modifying the E protein, attenuated live vaccines for various flavivirus infections can be brought into practical application in a short time, without reducing the immune induction potential.
Brief Description of the Drawings
Figure 1 shows the differences in the nucleotide sequence of genomic cDNA and the amino acid sequence of polyprotein between the Japanese encephalitis virus L-17 strain and JaOH0566 strain. Nt represents nucleotide; AA repersents amino acid. Amino acid positions are shown by number, as counted from the amino acid next to the starting methionine of the polyprotein.
Figure 2 shows the results of a comparison of amino acids that have mutated from the parent strain (JaOH0566 strain) (indicated by bold letters) in the polyprotein of the Japanese encephalitis virus ML-17 strain, with the corresponding amino acids in the polyproteins of the JaOH0566 strain, JaOArS982 strain, JaGArOl strain, Nakayama strain, Beijing strain, SA14 strain, and SA14-14-2 strain. Amino acid positions are shown by number, as counted from the amino acid next to the starting methionine of the polyprotein. * represents that the amino

acid at the indicated position is the same as that of the ML-17 strain.
Figure 3 shows an outline of the method of preparing the full-length cDNA of the recombinant Japanese encephalitis virus MS-14 strain and MS-15 strain by the Long-PGR method.
Figure 4 shows an outline of the method of preparing the cDNA of the ML-17/WN (E gene) chimeric flavivirus by the Long™ PCR method.
Best Mode for Erobodying the Invention
The present invention provides a nucleic acid molecule comprising nucleotide sequences that encode the C protein, prM protein and NS protein of Japanese encephalitis virus (a first flavivirus), and a nucleotide sequence that encodes the E protein of a second flavivirus. Preferably, the present invention provides a nucleic acid molecule comprising a nucleotide sequence of the 5' untranslated region, the nucleotide sequences that encode the C protein, prM protein and NS protein, and a nucleotide sequence of the 3' untranslated region of Japanese encephalitis virus, and the nucleotide sequence that encodes the E protein of the second flavivirus. Furthermore, this nucleic acid molecule comprises nucleotide mutations that produce one or more amino acid mutations capable of attenuating the virus as described below, in the nucleotide sequence(s) that encode(s) the prM protein and/or NS protein of Japanese encephalitis virus.
The present invention also provides a chimeric flavivirus encoded by such a nucleic acid molecule.
In this specification, "a nucleic acid molecule" means a single-stranded or double-stranded DNA or RNA.
In this specification, Ma nucleotide sequence" means a sequence of deoxyribonucleotide (shown by A, G, C, and T) or a sequence of ribonucleotide (shown by A, G, C, and U) unless otherwise specified.
In this specification, the left end represents the 5'

terminus and the right end represents the 3' terminus for a single-stranded nucleotide sequence; the left end represents the N terminus (amino terminus) and the right end represents the C terminus (carboxyl terminus) for an amino acid sequence unless otherwise specified.
In this specification, amino acids are shown using 1-letter abbreviation or 3-letter abbreviation in the Standard denotation system for amino acids unless otherwise specified.
In this specification, "attenuated" means that a virus is low virulent (low toxic) such that the virus can be used safely as a Vaccine for an animal subject to vaccination {for example, human and non-human mammals (for example, monkeys, horses, cattle, sheep, swine, dogs, cats, rabbits, rats, mice and the like), and birds and the like}.
In this specification, "the virus can be used (safely) as a Vaccine" means that growth of the virus is observed at the site of inoculation of the Vaccine but ends without manifesting serious Symptoms, and that specific immunity is conferred which prevents the onset of a disease caused by a highly virulent virus inoculated in a subsequent challenge test with the virus against an individual inoculated with the Vaccine.
Japanese encephalitis virus used as a first flavivirus to prepare the chimeric flavivirus of the present invention is not subject to limitation, as long as the prM protein and/or NS protein of the chimeric flavivirus of the present invention finally comprises one or more amino acid mutations that attenuate the virus as described below.
Therefore, any strain may be used out of the various strains of Japanese encephalitis virus (for example, ML-17 ■ strain, JaOH0566 strain, JaOArS982 strain, JaGArOl strain, Nakayama strain, Beijing strain, SA14 strain, SA14-14-2 strain and the like).
The genomes of various Japanese encephalitis virus strains have been cloned, and the complete or partial

nucleotide sequences thereof have been determined. See, for example, Sumiyoshi et al., Virology 161:4 97-510, 1987 for the JaOArS982 strain; McAda et al., Virology 158:348-360, 1987 for the Nakayama strain; Hashimoto et al., Virus Genes 1:305-317, 1988 for the Beijing strain; Nitayaphan et al., Virology 177:541-552, 1990 for the SA14 strain and SA14-14-2 strain.
Information on the genome sequences of various Japanese encephalitis viruses can also be obtained from publicly accessible gene databases such as GenBank. See, for example, GenBank accession number: M18370 for the JaOArS982 strain; GenBank accession number: AF069076 for the JaGArOl strain; GenBank accession number: L48961 for the Beijing-1 strain; GenBank accession number: M55506 for the SA14 strain; GenBank accession number: AF315119 for the SA14-14-2 strain.
To prepare a nucleic acid molecule comprising a nucleotide sequence that encodes the chimeric virus of the present invention, a nucleic acid molecule comprising a nucleotide sequence that encodes Japanese encephalitis virus is prepared, and can be used as the gene backbone for chimeric flavivirus. As examples of the nucleic acid molecule comprising a nucleotide sequence that encodes Japanese encephalitis virus, genomic RNA, cDNA, synthetic RNA, synthetic DNA and the like can be mentioned.
Alternatively, to prepare a nucleic acid molecule comprising a nucleotide sequence that encodes the chimeric flavivirus of the present invention, any fragment (for example, PCR-amplified DNA fragment and the like) of a nucleic acid molecule comprising a nucleotide sequence that encodes Japanese encephalitis virus can be used.
The genomic RNA of Japanese encephalitis virus can be prepared by a commonly known method from cells (for example, MMC-LK2 cells, HeLa cells, N2a cells, PS cells, BSC-1 cells, HL-CZ cells, LLC-MK2 cells, Vero cells, BHK cells, mosquito-derived C6/36 cells, mouse- or hamster-derived intracerebral

:ells), growing chicken eggs and the like infected with Japanese encephalitis virus. Japanese encephalitis virus strain used to prepare a genomic RNA is not subject to Limitation; for example, the strains as listed above can be ased.
A cDNA of Japanese encephalitis virus can be constructed froiti a genomic RNA according to a commonly known method (for example, the method described in Sumiyoshi et al., Virology 161:497-510, 1987).
Alternatively, a genomic RNA or cDNA of Japanese encephalitis virus or any fragment thereof can also be chemically synthesized on the basis of genome sequence Information on Japanese encephalitis virus by a commonly known method.
The genomic RNA or cDNA of Japanese encephalitis virus or any fragment thereof can be amplified with a genomic RNA or cDNA as the template by Polymerase Chain Reaction (abbreviated as "PCR method"), Reverse Transcriptase-Polymerase Chain Reaction (abbreviated as "RT-PCR method"), Long Polymerase Chain Reaction (abbreviated as "Long-PCR method"), and/or Long-Reverse Transcriptase-Polymerase Chain Reaction (abbreviated as "Long-RT-PCR method").
Alternatively, a genomic RNA or cDNA of Japanese encephalitis virus or any fragment thereof can also be inserted to a vector and cloned.
As examples of the vector used, plasmids such as pBR322, pBR325, pBR327, pBR328, pUC7, pUC8, püC9, pCU18, pUC19, pHSG298, pHSG299, pSCIOl, pGBM5, and pCRII can be mentioned- As the cloning vector, bacteriophage, cosmid, phagemid and the like can also be used, These cloning vectors are commercially available from, for example, NIPPON GENE CO., LTD. and the like.
As the second flavivirus used in the present invention, a flavivirus other than Japanese encephalitis virus, for example, West Nile virus, denguel-4 virus, yellow fever virus, St. Louis

prepared, and a region that encodes the E protein thereof can be used. As examples of the nucleic acid molecule comprising a nucleotide sequence that encodes a second flavivirus, genomic RNA, cDNA, synthetic RNA, synthetic DNA and the like can be » mentioned.
Alternatively, to prepare a nucleic acid molecule comprising a nucleotide sequence that encodes the chimeric flavivirus of the present invention, any fragment (for example, PCR-amplified DNA fragment and the like) of a nucleic acid ' molecule comprising a nucleotide sequence that encodes a second flavivirus can be used.
The genomic RNA of a second flavivirus can be prepared by the same method as that for Japanese encephalitis virus.
A cDNA of a second flavivirus can also be constructed from a genomic RNA by the same technique as that for Japanese encephalitis virus.
Alternatively, a genomic RNA or cDNA of a second flavivirus or any fragment thereof can also be chemically synthesized on the basis of commonly known genome sequence Information on the virus used as the second flavivirus, by a commonly known method.
The genomic RNA or cDNA of a second flavivirus or any fragment thereof can be amplified by the PCR method, RT-PCR method, Long-PCR method and/or Long-RT-PCR method, with a genomic RNA or cDNA as the template, as in Japanese encephalitis virus.
Alternatively, a genomic RNA or cDNA of a second flavivirus or any fragment thereof can also be inserted to an appropriate vector as listed above and cloned, as in Japanese encephalitis virus.
A nucleic acid molecule (DNA or RNA) comprising a nucleotide sequence that encodes the chimeric flavivirus of the present invention is prepared by replacing a nucleotide sequence that encodes the E protein in a nucleic acid molecule

comprising a nucleotide sequence that encodes Japanese encephalitis virus with a nucleotide sequence that encodes the envelop protein of a second flavivirus.
Replacement of the region that encodes the E protein in a nucleic acid molecule comprising a nucleotide sequence that encodes Japanese encephalitis virus can be performed by a commonly known recombinant technology (for example, the method utilizing the Long-PCR method described in Morita et al., Virology 287:417-426, 2001, and the like).
Alternatively, a nucleic acid molecule (DNA or RNA) comprising a nucleotide sequence that encodes the chimeric flavivirus of the present invention can also be prepared by chemical synthesis by designing the entire nucleotide sequence that encodes the chimeric flavivirus of the present invention, on the basis of genome sequence Information on Japanese encephalitis virus and a second flavivirus.
When a nucleic acid molecule comprising a nucleotide sequence that encodes the chimeric flavivirus of the present invention is prepared as DNA, a promoter sequence for in vitro transcription is introduced to the 5' terminus of the DNA, The DNA that encodes the chimeric flavivirus of the present invention is transcribed to RNA by an RNA Polymerase corresponding to the promoter introduced, at any stage prior to expression of the chimeric flavivirus protein. As examples of the promoter sequence used, the T7 RNA Polymerase promoter, SP6 RNA Polymerase promoter and the like can be mentioned.
The RNA that encodes the chimeric flavivirus of the present invention is introduced by a commonly known gene introduction technology in the art such as transfection, electroporation, or microinjection, into cells suitable for protein expression (for example, C6/36 cells, Vero cells, BHK cells, MMC-LK2 cells, HeLa cells, N2a cells, PS cells and the like), and the chimeric flavivirus protein is expressed in these cells.

Alternatively, for the chimeric flavivirus of the present invention, the chimeric flavivirus protein can be obtained by utilizing a method of protein production not using a cell System, wherein a genetic Information translation System for the organism is provided in a test tube by the addition of a Substrate, enzyme and the like to cell homogenate or extract (also referred to as cell-free system expression; see, for example, US Patent No. 5,478,730; Madin et al., Proc. Natl. Acad. Sei. USA 97:559-564, 2000; Sawasaki et al., Proc. Natl. Acad. Sei. USA 99:14652-14657, 2002).
The nucleic aeid molecule comprising a nucleotide sequence that encodes the chimeric flavivirus of the present invention comprises a nucleotide mutation that produces one or more amino aeid mutations that attenuate the virus in the nucleotide sequence that encodes the prM protein and/or NS protein derived from Japanese encephalitis virus in this nucleic aeid molecule.
In detail, when expressed with the amino aeid sequences of the individual structural proteins and non-structural proteins of the Japanese encephalitis virus JaOArS982 strain (see H. Sumiyoshi et al., Virology 161:497-510, 1987) as a reference, the amino aeid mutations that attenuate the virus are the following amino aeid substitutions: Substitution of the Ist methionine of the prM protein by isoleucine; Substitution of the 148th asparagine of the prM protein (56th of M protein) by threonine; Substitution of the 4th alanine of the NS2A protein by serine; Substitution of the 51st asparagine of the NS4B protein by lysine; Substitution of the 52nd valine of the NS4B protein by isoleucine; Substitution of the 68th threonine of the NS4B protein by serine; Substitution of the 126th leucine of the NS5 protein by methionine; and/or Substitution of the 854th serine of the NS5 protein by asparagine.
Alternatively, the above-described amino aeid substitutions can also be performed using conservative amino

acids for the amino acids introduced at the respective amino acid positions.
In this specification, "conservative amino acids" mean amino acids similar to each other in terms of physicochemical properties; examples thereof include amino acids classified under the same group, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gin, Asn), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids having a hydroxyl group (Ser, Thr) and amino acids having a small side-chain (Gly, Ala, Ser, Thr, Met).
Preferably, the chimeric flavivirus of the present invention comprises at least Substitution of the Ist methionine of the prM protein by isoleucine and/or Substitution of the 148th asparagine of the prM protein (56th of M protein) by threonine, out of the above-described amino acid substitutions.
The above-described amino acid mutations can be introduced in any step for preparing a nucleic acid molecule comprising a nucleotide sequence that encodes the chimeric flavivirus of the present invention.
Introduction of the above-described amino acid mutations can be performed using, for example, the site-directed mutagenesis method utilizing the Long-PCR method, described in Morita et al., Virology 287:417-426, 2001, or by applying a commonly known method such as the Kunkel method or the gapped duplex method or a mutagenesis kit utilizing these methods (available from, for example, Takara Bio Inc.) and the like to a cDNA cloned to plasmid.
Alternatively, these mutations can be introduced to the chimeric flavivirus of the present invention by using a nucleic acid molecule comprising a nucleotide sequence that encodes a Japanese encephalitis virus having one or more amino acid mutations that attenuate the virus in the prM and/or NS protein to prepare a nucleic acid molecule comprising a nucleotide

sequence that encodes the chimeric flavivirus of the present invention.
A nucleic acid molecule comprising a nucleotide sequence that encodes a Japanese encephalitis virus having one or more amino acid mutations that attenuate the virus in the prM and/or NS protein can be prepared by artificially introducing these mutations to a genomic RNA or cDNA of a Japanese encephalitis virus strain not containing these mutations (for example, JaOH0566 strain, JaOArS982 strain, JaGArOl strain, Nakayama strain, Beijing strain, SA14 strain, SA14-14-2 strain and the like), using the aforementioned commonly known method of site-directed mutagenesis.
A recombinant Japanese encephalitis virus can be prepared by expressing a virus protein from the nucleic acid molecule comprising a nucleotide sequence that encodes a Japanese encephalitis virus having one or more amino acid mutations that attenuate the virus in the prM and/or NS protein, thus artificially prepared, by a commonly known method as described above.
By infecting this recombinant Japanese encephalitis virus to suitable cells as described above, and culturing the cells, a large amount of genomic RNA can be prepared from the cultured cells-
Furthermore, this recombinant Japanese encephalitis virus is an attenuated Japanese encephalitis virus per se, and can be a promising candidate for an attenuated live Vaccine for Japanese encephalitis.
A nucleic acid molecule comprising a nucleotide sequence that encodes a Japanese encephalitis virus having one or more amino acid mutations that attenuate the virus in the prM and/or NS protein can also be prepared by acquiring a genomic RNA or cDNA from a Japanese encephalitis virus mutant strain natively comprising one or more such amino acid mutations.
As examples of the Japanese encephalitis virus mutant

strain natively comprising one or more amino acid mutations that attenuate the virus in the prM and/or NS protein, the Japanese encephalitis virus ML-17 strain can be mentioned. The ML-17 strain is a Japanese encephalitis virus Vaccine strain for swine (see, for example, Yoshida et al., BIKEN JOURNAL 24:47-67, 1981), and is available from The Research Foundation for Microbial Diseases of Osaka University (based in Osaka University, 3-1, Yamadaoka, Suita-shi, Osaka).
Alternatively, from among mutant strains of Japanese encephalitis virus resulting from passage in cells and the like, a strain having one or more amino acid mutations that attenuate the virus in the prM and/or NS protein may be selected by a commonly known method, and a genomic RNA or cDNA thereof can also be used to prepare the chimeric flavivirus of the present invention.
The nucleic acid molecule (genomic RNA or cDNA) comprising a nucleotide sequence that encodes a Japanese encephalitis virus having one or more amino acid mutations that attenuate the virus in the prM and/or NS protein, thus prepared, may be inserted to a cloning vector as described above after being fragmented as required. For example, utilizing such a recombinant vector, the chimeric flavivirus of the present invention can be prepared safely and conveniently.
Construction of the chimeric flavivirus of the present invention can be confirmed by, for example, acquiring the gene from the chimeric virus prepared, determining all or a portion of the base sequence thereof or corresponding amino acid sequence (for example, the portion where the mutation has been introduced, the Joint of the two viruses and the like), and confirming that the sequence matches with the intended sequence.
The present invention also provides an attenuated live Vaccine comprising the chimeric flavivirus of the present invention (hereinafter also referred to as the Vaccine of the present invention). Because the E protein of virus contains an

antigenic determinant that induces a neutralizing antibody, inoculation of the chimeric flavivirus of the present invention as a Vaccine results in the production of an antibody against the second flavivirus species used as the E protein.
To prevent various flavivirus infections, the Vaccine of the present invention can be inoculated to, for example, animals such as humans and non-human mammals (for example, monkeys, horses, cattle, sheep, swine, dogs, cats, rabbits, rats, mice and the like), and birds.
The Vaccine of the present invention can be produced in the form of a Suspension or freeze-dried preparation. The Vaccine of the present invention can comprise, in addition to the chimeric flavivirus of the present invention, a pharmaceutically acceptable stabilizer (for example, sucrose, lactose, glucose, gelatin, gelatin hydrolysates, sodium L-glutamate, serum albumin and the like), soothing agent (for example, glucose and the like) and the like in common use in Vaccine preparations.
Upon vaccination, the Vaccine of the present invention can normally be dissolved or suspended in a pharmaceutically acceptable carrier. As examples of the carrier, liquid carriers such as water, saline (including physiological saline), and buffer Solutions (for example, phosphate buffer Solution) can be mentioned.
The Vaccine of the present invention is representatively prescribed as a sterile aqueous Solution containing 102 to 106 PFU of the chimeric flavivirus of the present invention per a dose of 0.1 to 1.0 ml, and can be inoculated, for example, subcutaneously, intradermally, intramuscularly and the like.
Alternatively, because some flaviviruses are known to infect via the mucosa, the Vaccine of the present invention may be administered orally or transnasally according to the second flavivirus species chosen.
Furthermore, a nucleic acid molecule (RNA or DNA)

comprising a nucleotide sequence that encodes the chimeric flavivirus of the present invention per se can also be used as a nucleic acid Vaccine preparation.
Confirmation of the efficacy and safety of the chimeric virus constructed can be performed by, for example, evaluating brain neurotoxicity, infectivity from peripheral to the central nervous System (brain nerve invasiveness), infection-preventing effect, presence or absence of viremia, neutralizing antibody production and the like in animals (for example, micef monkeys and the like) by a commonly known method of evaluation in the art.
The present invention is hereinafter described in more detail by means of the following Examples, which, however, are for illustrative purposes only and never limit the scope of the invention.
Examples
(Example 1)
(Determination of attenuating mutation sites of the Japanese
encephalitis virus Vaccine ML-17 strain)
To determine the attenuating mutation sites of the Japanese encephalitis virus Vaccine ML-17 strain, first, the nucleotide sequence of full-length genomic cDNA and the amino acid sequence of polyprotein were compared between the ML-17 strain and its parent strain, i.e., the wild-type virulent Japanese encephalitis virus JaOH0566 strain.
The nucleotide sequence of full-length genomic cDNA of the ML-17 strain (obtained from The Research Foundation for Microbial Diseases of Osaka University) (SEQ ID N0:1) and the nucleotide sequence of full-length genomic cDNA of the JaOH0566 strain (SEQ ID NO:3) were determined according to the method disclosed in Sumiyoshi et al.f Virology 161:497-510, 1987. For both strains, the cDNAs were constructed using the PCR primers shown in Table 1A, and the nucleotide sequences of the cDNAs constructed were determined using the primers for sequencing

shown in Table IB. Furthermore, the amino acid sequence of the polyprotein of the ML-17 strain (SEQ ID NO:2) and the amino acid sequence of the polyprotein of the JaOH0566 strain (SEQ ID NO:4) were deduced from the nucleotide sequences of the respective full-length genomic cDNAs.

*: F represents a forward primer; R represents a reverse primer.
As a result of a comparison of the nucleotide sequences of the genomic cDNAs, it was found, as shown in Figure 1, that the ML-17 strain had 25 nucleotide substitutions, with 10 amino acid substitutions produced due to 10 of these nucleotide substitutions, at the 127th, 274th, 1209th, 2462nd, 2463rd 2479th, 2652nd, 2751st, 2896th, and 3380th amino acids, as counted from the.amino acid next to the starting methionine of the polyprotein.
Furthermore, the above-described amino acid mutations in the polyprotein of the ML-17 strain were compared with the amino acid sequences of the corresponding sites of the polyproteins of the JaOArS982 strain, JaGArOl strain, Nakayama strain, Beijing strain, and SA14 strain, which are other wildtype virulent Japanese encephalitis viruses, and the SA14-14-2 strain, which is another Japanese encephalitis virus Vaccine.
The putative amino acid sequences of the polyproteins of the following strains were obtained from GenBank and used in this Example:
Amino acid sequence of the polyprotein of the JaOArS982 strain (GenBank accession number: M18370);
Amino acid sequence of the polyprotein of the JaGArOl strain (GenBank accession number: AF069076); Amino acid sequence of the polyprotein of the Beijing-1 strain

(GenBank accession number: L48961);
Amino acid sequence of the polyprotein of the SA14 strain (GenBank accession number: M55506);
Amino acid sequence of the polyprotein of the SA14-14-2 strain (GenBank accession number: AF315119).
For the amino acid sequence of the polyprotein of the Nakayama strain, the amino acid sequence of the region necessary for the comparison was obtained from the gene and/or amino acid partial sequence Information described in GenBank accession number: AF112297; McAda et al., Virology. 158(2):348-60, 1987 and the like, and used in this Example.
As shown in Figure 2, as a result of a comparison of the amino acid sequences of the polyproteins, a total of eight amino acid substitutions at the 127th, 274th, 1209th, 2462nd, 2463rd, 2479th, 2652nd, and 3380th amino acid positions, as counted from the amino acid next to the starting methionine of the polyprotein, out of the above-described 10 amino acid substitutions between the ML-17 strain and the JaOH0566 strain, were found to be amino acid mutations intrinsic to the ML-17 strain.
From a comparison with the positions in the individual structural proteins and non-structural proteins in the polyprotein of the Japanese encephalitis virus (JaOArS982 strain) described in Sumiyoshi et al., Virology 161:497-510, 1987, it was found that the positions of these eight amino acid substitutions in the polyprotein corresponded to the Ist and 148th amino acids of the prM protein (56th of M protein); the 4th amino acid of the NS2A protein; the 51st, 52nd, and 68th amino acids of the NS4B protein; and the 126th and 854th amino acids of the NS5 protein, respectively.
(Example 2)
(Preparation of recombinant Japanese encephalitis virus MS-14
strain and MS-15 strain)

To confirm the effects of amino acid mutations intrinsic to the ML-17 strain on the attenuation of the virus, according to the site-directed mutagenesis method utilizing the Long-PCR method, described in Morita et al.r Virology 287:417-426, 2001, using the gene of the wild-type virulent Japanese encephalitis virus JaOArS982 strain as the backbone, MS-14 strain, which is a recombinant Japanese encephalitis virus incorporating a nucleotide mutation that produces Substitution of the Ist methionine of the prM protein by isoleucine (Substitution of G by A at position 479 of the genome), and MS-15 strain, which is a recombinant Japanese encephalitis virus incorporating a nucleotide mutation that produces Substitution of the 148th asparagine of the prM protein (56th of the M protein) by threonine (Substitution of A by C at position 919 of the genome), were prepared. (Preparation of the MS-14 strain)
As shown in Figure 3, with the gene RNA of Japanese encephalitis virus (JaOArS982 strain) as the template, using a set of primer 1 and primer 2, a set of primer 3 and primer 4, and a set of primer 5 and primer 6, by the Long-RT-PCR method, Japanese encephalitis virus gene fragments corresponding to the respective primer sets (fragments 1, 2, and 3) were prepared.
Furthermore, after each fragment was purified by agarose electrophoresis, first, with fragments 1 and 2 as the templates, using a set of primers 1 and 4, again by the Long-PCR method, gene fragment 4 was prepared. This gene fragment was purified in the same manner; next, with gene fragments 3 and 4 as the templates, using a set of primers 1 and 6, the Long-PCR method was performed to prepare a full-length Japanese encephalitis virus cDNA having the T7 promoter sequence at the 5' terminus thereof (fragment 5).
With this full-length Japanese encephalitis virus cDNA (fragment 5) as the template, an in vitro RNA synthesis reaction was performed to prepare an artificial full-length

Japanese encephalitis virus gene RNA. This RNA was introduced into mosquito cells (C6/36 cells) by the electroporation method and the cells were cultured for 5 days, after which the recombinant virus (MS-14 strain) emerging in the culture supernatant was recovered. (Preparation of the MS-15 strain)
For the MS-15 strain, the recombinant virus was recovered according to the above-described method except that the recombinant virus was prepared using primer 7 instead of primer 2, and primer 8 instead of primer 3.
Furthermore, for control, a mutation-free virus was acquired using primer 9 instead of primer 2, and primer 10 instead of primer 3.
The primers used in this Example are shown in Table 2.

represents a complementary sequence. The underline represents the T7 promoter sequence; the double underline represents a nucleotide mutation.
(Example 3)
(Evaluation of the growing potentials of the recombinant Japanese encephalitis virus MS-14 strain and MS-15 strain in BHK cells)
The growing potentials of the recombinant Japanese encephalitis virus MS-14 stra in and MS—15 strain prepared in Example 2 in BHK cells were evaluated. For control, the wildtype virulent Japanese encephalitis virus JaOArS982 strain was used.
Each of the MS-14 strain, MS-15 strain, and JaOArS982 strain was infected to BHK cells; 24, 48, and 72 hours after infection, cell culture supernatants were collected and checked for the emergence of virus. The virus contents in the culture supernatants at each time were measured by the plaque method. For all strains, growth in the BHK cells was confirmed.
(Example 4)
(Evaluation of the neurotoxicity of the recombinant Japanese encephalitis virus MS-14 strain and MS-15 strain in normal mature mice)
Using the recombinant Japanese encephalitis virus MS-14 strain and MS-15 strain prepared in Example 2, the neurotoxicity of each strain was evaluated by the intraperitoneal inoculation method in normal mature mice. For virulent control, the wild-type virulent Japanese encephalitis virus JaOArS982 strain was used; for attenuated control, the Japanese encephalitis virus Vaccine ML-17 strain was used.
Ten mice (4-week-old male and female ICR mice, 5 animals in each group) were given 101 to 106 plaque forming units of the MS-14 strain, MS-15 strain, ML-17 strain, or JaOArS982

strain oy mtracerebral inoculation, and observed daily for 3 weeks, and LD50 was calculated by the Read-Muench method. The results of this test are shown in Table 3.

*: MS-14 rev represents a revertant of MS-14, prepared from MS-14 using Long-PCR-based site-directed mutagenesis. > As shown in Table 3, the MS-14 strain and MS-15 strain were significantly attenuated compared to the virulent JaOArS982 strain. Particularly, the MS-14 strain had a toxicity similar to that of the ML-17 strain, which is a Japanese encephalitis virus Vaccine strain.
This demonstrated that neurotoxicity in mice could be reduced by amino acid Substitution in the prM protein.
From the results above, it was suggested that amino acid mutations introduced to the MS-14 strain and MS-15 strain, which are recombinant Japanese encephalitis viruses, might be useful in preparing a Japanese encephalitis live Vaccine. Furthermore, because MS-14 exhibits a low degree of pathogenicity in high-dose inoculation, it was suggested that a mutation on NS might also be necessary for attenuation.
(Example 5)
(Construction of cDNA of chimeric flavivirus of Japanese
encephalitis virus and West Nile virus (ML-17/WN (E gene)))

To prepare ML-17/WN (E gene), which is a chimeric flavivirus wherein the E protein of the Japanese encephalitis virus Vaccine ML-17 strain is replaced by the E protein of West Nile virus, a cDNA of ML-17/WN (E gene) was constructed in accordance with the method of preparing a recombinant Japanese encephalitis virus utilizing the Long-PCR method, described in Morita et al., Virology 287:417-426,2001.
As shown in Figure 4, with the gene RNA of a Japanese encephalitis virus live Vaccine strain (ML-17 strain) as the template, using a set of primer 1A and primer 2A and a set of primer 5A and primer 6A, by the Long-RT-PCR method, Japanese encephalitis virus gene fragments corresponding to the respective primer sets (fragments 1A and 3A) were prepared.
Also, with the gene RNA of West Nile virus (NY99-35262-11 strain) as the template, using a set of primer 3A and primer 4A, by the Long-RT-PCR method, West Nile virus gene fragment 2A was prepared.
After each fragment was purified by agarose electrophoresis, first, with fragments 1A and 2A as the templates, using a set of primers 1A and 4A, again by the Long-PCR method, gene fragment 4A was prepared.
This gene fragment was purified in the same manner; then, with gene fragments 3A and 4A as the templates, using a set of primers 1A and 6A, the Long-PCR method was performed to prepare a chimeric virus cDNA (fragment 5A) of ML-17/WN (E gene), which has the T7 promoter sequence at the 5' terminus thereof and the West Nile virus E protein gene.
The primers used in this Example are shown in Table 4.

*: JE represents the Japanese encephalitis virus JaOAr982 strain; ML17 represents the sequence of the Japanese encephalitis virus ML-17 strain; WN represents the sequence of West Nile virus. S or N represents a sense sequence; R represents a complementary sequence. The underline indicates the T7 promoter sequence.
(Example 6)
(Production of chimeric flavivirus from cDNA of ML-17/WN (E gene))
With the cDNA of the chimeric flavivirus of Japanese encephalitis virus and West Nile virus, constructed in Example 5 (fragment 5A in Figure 4), as the template, an in vitro RNA synthesis reaction was performed to prepare an artificial full-length chimeric virus gene RNA. This RNA was introduced into mosquito cells (C6/36 cells) by the electroporation method and the cells were cultured for 5 days, after which the chimeric virus emerging in the culture supernatant was recovered.

(Example 7)
(Evaluation of the growing potential of ML-17/WN (E gene)
chimeric flavivirus)
The chimeric virus used was the ML-17/WN (E gene) as obtained in Example 6. ML-17/WN (E gene) and the ML-17 strain were grown in C6/36 cells and BHK cells, and the infected
culture broths were dispensed and stored at -70°C. For both types of cells, an Eagle MEM culture broth supplemented with non-essential amino acids was used, with fetal bovine serum added at a concentration as required (2 to 10%) . The infectivity titers of the culture broths in the growth experiment are shown in Table 5. ML-17/WN (E gene) had a growing potential equivalent to that of ML-17.
Table 5 Growth of ML-17/WN (E gene) chimeric flavivirus and ML-17 in C6/36 cells and BHK cells

(Example 8)
(Evaluation of the neurotoxicity of ML-17/WN (E gene) chimeric virus)
Using the recombinant chimeric virus ML-17/WN (E gene) prepared in Example 6, the neurotoxicity was evaluated by the intracerebral inoculation method in normal mature mice. Also examined by intraperitoneal administration was infection from peripheral to the central nervous System (brain nerve invasiveness). For control, the JaOH0566 strain, which is a wild-type highly virulent strain Japanese encephalitis viruses, the West Nile virus NY99-3562-11 strain, and the Japanese

encephalitis virus Vaccine ML-17 strain, which is an attenuated strain, were used.
C57BL/B6 mice at 4 weeks after birth were given 101 to 107 of each virus by intracerebral inoculation, and observed for 28 days. LD5o was calculated by the Read-Muench method.
Table 6 shows the LD5o of ML-17/WN (E gene) obtained from the experiment. ML-17/WN (E gene) was significantly attenuated compared to the highly virulent JaOH0566 strain. Particularly, ML-17/WN (E gene) had a toxicity similar to that of the ML-17 strain, which is a Japanese encephalitis virus Vaccine strain.

(Example 9)
(Property of the ML-17/WN (E gene) chimeric virus)
The infectivity of the ML-17/WN (E gene) chimeric virus to mosquitoes was investigated. Culex tritaeniorhynchus 0K7 was allowed to suck a mixture of the virus in rabbit blood
(antibody negative) by the transmembrane method. By evaluating the PFÜ (Plaque-Forming Unit) and the incidence rate in suckling mice, the infection rate was calculated.
After the mosquitoes allowed to suck the chimeric virus by the transmembrane method were reared for 10 days and 21 days, they were emulsified, and demonstration of the presence of the virus was attempted by intracerebral inoculation in suckling mice and PFU evaluation. Even when the ML-17/WN (E gene) virus was allowed to be sucked at not less than 102 PFU per mosquito, there was absolutely no evidence for the presence of the virus. The ML-17/WN (E gene) chimeric virus did not exhibit

susceptibsility to Culex tritaeniorhynchus.
(Example 10)
(Evaluation of the defensive potency of a live Vaccine comprising the ML-17/WN (E gene) chimeric virus)
An immunization experiment was performed using 2-week-old C57BL/B6 and C3H/He mice.
An investigation was performed using the two inbred lines of mice. The non-immunized control group was fatally infected 1 by an intracerebral challenge with West Nile virus (NY99-35262-11 strain), whereas the group given ML-17/WN (E gene) by intraperitoneal inoculation exhibited significant defense agäinst infection.
Industrial Applicability
According to the present invention, an attenuated chimeric flavivirus having attenuating mutation(s) in portion(s) other than the E protein is provided. Because the attenuation of the chimeric flavivirus of the present invention can be achieved without modifying the E protein, attenuated live vaccines for various flavivirus infections can be brought into practical application in a short time, without reducing the immune induction potential.
This application is based on a patent application No. 2004-374630 filed in Japan, the contents of which are entirely incorporated herein by reference.

Claims
1. A nucleic acid molecule comprising nucleotide sequences that
encode the capsid protein, pre-membrane protein and non-
structural protein of Japanese encephalitis virus, and a
nucleotide sequence that encodes the envelop protein of a
second flavivirus,
wherein the nucleotide sequence(s) that encode(s) the pre-membrane protein and/or non-structural protein of Japanese encephalitis virus comprise(s) nucleotide mutations that produce one or more amino acid mutations that attenuate the virus -
2. The nucleic acid molecule of claim 1, wherein the Japanese encephalitis virus is the ML-17 strain.
3. The nucleic acid molecule of claim 1 or 2, wherein the second flavivirus is selected from the group consisting of West Nile virus, dengue 1-4 virus, yellow fever virus, St. Louis encephalitis virus, tick-borne encephalitis virus, Kunjin virus, Central European encephalitis virus, Kyasanur Forest virus, Murray Valley encephalitis virus, Omsk hemorrhagic fever virus, Powassan virus, Russian spring-summer encephalitis virus, Yokose virus, Apoi virus, and Aroa virus.
4. An attenuated chimeric flavivirus encoded by the nucleic acid molecule of any one of Claims 1 to 3.
5. An attenuated live Vaccine comprising the attenuated
i chimeric flavivirus of claim 4.
6. A method of preparing the nucleic acid molecule of claim 1,
which comprises the following steps:
a step for replacing a nucleotide sequence that encodes the

envelop protein in a nucleic acid molecule comprising a nucleotide sequence that encodes Japanese encephalitis virus with the nucleotide sequence that encodes the envelop protein of the second flavivirus; and
a step for introducing nucleotide mutations that produce one or nore amino acid mutations that attenuate the virus into the nucleotide sequence(s) that encode(s) the pre-membrane protein and/or non-structural protein of Japanese encephalitis virus.
7. A method of preparing the nucleic acid molecule of claim 1,
which comprises the following step:
a step for replacing a nucleotide sequence that encodes the envelop protein in a nucleic acid molecule comprising a nucleotide sequence that encodes a Japanese encephalitis virus having one or more amino acid mutations that attenuate the virus in the pre-membrane protein and/or non-structural protein with the nucleotide sequence that encodes the envelop protein of the second flavivirus.
8. A method of preparing an attenuated chimeric flavivirus, which comprises a step for expressing chimeric flavivirus proteins from the nucleic acid molecule of any one of Claims 1 to 3.
9. A nucleic acid molecule comprising a nucleotide sequence that encodes a Japanese encephalitis virus having one or more amino acid mutations that attenuate the virus in the pre-membrane protein and/or non-structural protein.

10. A vector comprising the nucleic acid molecule of claim 9.
11. An attenuated Japanese encephalitis virus encoded by the nucleic acid molecule of claim 9.

12. A method of preparing the nucleic acid molecule of Claim 9, which comprises a step for introducing nucleotide mutations that produce one or more amino acid mutations that attenuate the virus into nucleotide sequence(s) that encode(s) the pre-membrane protein and/or non-structural protein in a nucleic acid molecule comprising a nucleotide sequence that encodes Japanese encephalitis virus.
13- A method of preparing an attenuated Japanese encephalitis virus, which comprises a step for expressing Japanese encephalitis virus proteins from the nucleic acid molecule of claim 9.